Flexibility and constraint: Evolutionary remodeling of the sporulation initiation pathway in Firmicutes

Abstract

The evolution of signal transduction pathways is constrained by the requirements of signal fidelity, yet flexibility is necessary to allow pathway remodeling in response to environmental challenges. A detailed understanding of how flexibility and constraint shape bacterial two component signaling systems is emerging, but how new signal transduction architectures arise remains unclear. Here, we investigate pathway remodeling using the Firmicute sporulation initiation (Spo0) pathway as a model. The present-day Spo0 pathways in Bacilli and Clostridia share common ancestry, but possess different architectures. In Clostridium acetobutylicum, sensor kinases directly phosphorylate Spo0A, the master regulator of sporulation. In Bacillus subtilis, Spo0A is activated via a four-protein phosphorelay. The current view favors an ancestral direct phosphorylation architecture, with the phosphorelay emerging in the Bacillar lineage. Our results reject this hypothesis. Our analysis of 84 broadly distributed Firmicute genomes predicts phosphorelays in numerous Clostridia, contrary to the expectation that the Spo0 phosphorelay is unique to Bacilli. Our experimental verification of a functional Spo0 phosphorelay encoded by Desulfotomaculum acetoxidans (Class Clostridia) further supports functional phosphorelays in Clostridia, which strongly suggests that the ancestral Spo0 pathway was a phosphorelay. Cross complementation assays between Bacillar and Clostridial phosphorelays demonstrate conservation of interaction specificity since their divergence over 2.7 BYA. Further, the distribution of direct phosphorylation Spo0 pathways is patchy, suggesting multiple, independent instances of remodeling from phosphorelay to direct phosphorylation. We provide evidence that these transitions are likely the result of changes in sporulation kinase specificity or acquisition of a sensor kinase with specificity for Spo0A, which is remarkably conserved in both architectures. We conclude that flexible encoding of interaction specificity, a phenotype that is only intermittently essential, and the recruitment of kinases to recognize novel environmental signals resulted in a consistent and repeated pattern of remodeling of the Spo0 pathway.

Distribution and preservation of the components of the engulfment. What is beyond representative genomes?

“…Considering this observation, we performed a test to measure the concurrency of the phyletic profile per genus, as described in the Methods section, to determine the prevalence of the studied proteins in the proteomes of the spore formers. The Spo0A response regulator was included in the phyletic profile since it has been mentioned as a sporulation signature of stage 0 in several publications [ 7 , 29 , 36 , 37 ], which is stage prior to engulfment. The results in Table 2 show the genus that presents phyletic profiles with a significant C-score in genera that have two or more species.…”

“…Since the phylum Firmicutes is polyphyletic [ 57 ] and the species are prone to horizontal gene transfer, the phylum taxonomy is continuously reorganized, especially the Clostridia class [ 62 , 63 ], which comprises various species of a gram stain-negative cell wall structure [ 64 ]. The aforementioned descriptions along with those that claim that the genes related to sporulation have been frequently lost throughout the evolution of Firmicutes [ 7 , 8 , 29 , 37 , 65 ], motivate to construct a Firmicutes species tree in which the organism selection was based on the organization of the phyletic profiles of the proteins involved in the engulfment and on the curated phenotypes, this means that a genus or a family may have more than one representative. We assume that a selection that uses species that have characteristic phyletic profiles, even if they belong to the same genus or family, will provide new clues that describe the evolutionary history of genes involved in the engulfment.…”

“…The establishment of the distribution and prevalence of genes among genomes is based on a comparative genomic analysis that predicts the presence of orthologs, such as those involved in sporulation [ 17 , 29 , 34 , 35 ]. A previous work of ours [ 36 ] and another by Davidson et al [ 37 ] based on the scanning of Hidden Markov Models profiles (HMM-profiles) in bacterial proteomes and the preservation of key genes in the genomic context have proven to be a powerful method to detect nearby and distant probable orthologs (POs). We tested this approach on the proteins of the phosphorelay initiating sporulation in Firmicutes [ 36 ].…”

Prediction of protein architectures involved in the signaling-pathway initiating sporulation in Firmicutes

“…Similar to the report of Davidson et al (PLoS Genet 14:1–33, 2018), we used Pfam profiles (El-Gebali et al in Nucleic Acids Res 47:D427–32, 2019) defining each protein and the genomic context surrounding the query gene to predict probable orthologs of the Spo0 pathway in Firmicutes. We present in this note a collection of 325 Firmicutes species organized by phylogenetic class and classified as spore formers, non-spore formers or unknown spore phenotype based on published literature, for which we predicted probable orthologs defining the signal transduction pathway initiating sporulation.…”

“…Comparative genomic analysis is a valuable tool to explore new genomes and metagenomes to search for functional processes, phylogenetic relationships, and evolutionary traits among organisms. An example of this comparative analysis is finding that proteins involved in the Spo0 pathway constitute of at least a sensor kinase, the phosphotransferases Spo0F and Spo0B, and the response regulator Spo0A [1], were suggested to be ancestral since it was found not only in Bacilli but also in some Clostridia [2]. While an original article from our group that describes the distribution and prevalence of the Spo0 pathway and also confirms that this pathway is ancestral was under review, the results mentioned above were published by Davidson and collaborators using a set 84 Firmicutes [2].…”